Phase Transition in Disease
Univ.-Prof. Dr. Ralf Weiskirchen Weiskirchen
Institut für Molekulare Pathobiochemie, experimentelle Gentherapie und Klinische Chemie
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Dr. Mirle Schemionek
Klinik für Hämatologie, Onkologie, Hämostaseologie und Stammzelltransplantation (Med. Klinik IV)
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Mesenchymal interactions and fibrogenic signalling in cancer development:
Molecular mechanisms in solid and hematologic neoplasms
Cancer deaths are a global burden that will continue to rise with an estimated 11.4 million people dying in 2030. Cancers can be solid such as hepatocellular carcinoma (HCC) that is one of the most vascular tumors or alternatively can be "liquid" such as in the case of leukemia. Although the phenotypic appearance of these different types of malignancies is rather diverse, it is most likely that there are common and specific mechanisms that are shared between the respective disease entities.
In particular, recent work has demonstrated that mesenchymal interaction and fibrogenic signaling are key drivers in setting a tumorigenic environment.
In this IZKF Joint Initiative, we have bundled the scientific activities in Aachen in the area "Oncology". We will foster the networking structure and publication output in this field to increase the international visibility. We attempt to make sustained progress in understanding the molecular differences between normal cells and cancer cells.
Therefore, we have set up a consortium in which 11 projects with contributing scientists/clinicians belonging to 12 different institutes/clinics work intensively together. Conceptual, the projects are focused on the environmental-driven pathogenesis using the example of HCC and the formation of blood cell tumor such as chronic myeloid leukaemia (CML).
The work programs of each subproject will be processed by combinations of researchers/clinicians coming from different institutes/clinics or alternatively from one research group that has already significant research expertise in the field of "Oncology" and substantial cooperations established in this field.
Here you find the summarys of the subprojects of the Oncology Research Area.
P01: Lipocalin 2 controls efferocytosis and formation of cancerassociated fibroblasts
PI: Univ.-Prof. Dr. rer. nat. Ralf Weiskirchen/Univ.-Prof. Dr. med. Steffen Koschmieder
Increased expression of lipocalin 2 (LCN2) has been observed in several cancers and was associated with aggressive tumor features, formation of distant metastatic spread and poor prognosis. It was demonstrated that LCN2 is critically required for leukemia induction in vivo, as shown by a lack of leukemia development in mice transplanted with Bcr-Abl transduced LCN2-deficient cells.
Also, during myelofibrosis, LCN2 acts as an inflammatory cytokine that contributes to the creation of a dysfunctional microenvironment, increased microvessel density, and osteosclerosis. In the liver, LCN2 is associated with the development of steatohepatitis by recruiting inflammatory neutrophils to the liver.
We have shown that hepatocytes are the major source for hepatic LCN2 that become induced by pro-inflammatory cytokines (e.g. IL-1β) via nuclear factor-κB (NF-κB) activation. LCN2 negatively modulates epithelial-to-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) through the epidermal growth factor or the transforming growth factor-β1/LCN2/Twist1 pathway suggesting that LCN2 prevents the generation of EMT-derived fibroblasts in cancerogenesis. In contrast, the opposite effect was observed in JAK2V617F-mutant murine and human hematopoietic cells, in which LCN2 stimulates formation of fibroblasts.
In the present project, we will
- analyse transcriptional changes of LCN2 expression during tumorigenesis in the liver and the hematopoietic system,
- unravel tumor-stromal interactions of LCN2 in the pathogenesis of respective tumors,
- determine the capacity of LCN2 in promoting immunosuppressive effects in the tumor microenvironment, and
- investigate the molecular activities and pathways by which LCN2 regulate tumor-initiating cell plasticity in blood and liver cells.
P02: Modulating macrophage-mediated microenvironmental remodelling to improve targeted anticancer therapy
PI: Univ.-Prof. Dr. rer. nat. Twan Lammers/Univ. Prof. Dr. med. Frank Tacke
Tumor-associated macrophages (TAM) comprise heterogeneous subsets, which play key roles in angiogenesis and in establishing a tumor-promoting microenvironment.
We here aim to understand and modulate macrophage-mediated microenvironmental tumor remodeling for more efficient targeted drug treatment of primary liver and metastatic breast cancer.
Our preliminary experimental evidence indicates that blocking CCR2+ macrophage infiltration contributes to a normalized vascular network (both in inflammatory liver disease and in tumors), potentially rendering the tumor stroma more accessible for targeted therapy.
In this project, we will characterize the composition and function of TAM subsets as well as their impact on tumor growth and stromal interactions. We will inhibit the infiltration of CCR2+ inflammatory monocytes via clinically tested anti-CCL2 RNA aptamers in mouse models of primary liver cancer (in a fibrotic liver, DEN+CCl4) and of metastatic breast cancer (4T1 model).
Moreover, we will systematically assess if anti-CCL2-based priming favors the accumulation, penetration and intratumoral distribution of drugs and drug delivery systems. These will include immunomodulating antibodies, as well as liposomes, polymers and micelles as potential drug carriers. The latter three will be loaded with the chemotherapeutic drug doxorubicin, which is clinically routinely used in patients suffering from liver and breast cancer.
Our efforts will provide pioneering proof-of-principle for combination therapies in which the fibrotic microenvironment in primary liver tumors and breast cancer metastases is primed with macrophage-modulating therapies to enable more efficient drug delivery and drug therapy.
P03: Epigenetic signatures of fibro-blasts in cancer
PI: Univ.-Prof. Dr. rer. nat. Dr. med. Wolfgang Wagner/Prof. Dr. Ivan G. Costa
Cancer-associated fibroblasts (CAFs) seem to have crucial impact on tumorigenesis and metastasis. However, it remains largely unknown how epigenetic programs are influenced in these non-malignant cells, and what cell type they originate from.
In this project we want to isolate mesenchymal stromal cells (MSC) from myeloproliferative neoplasms (MPN) and CAFs from hepatocellular carcinoma (HCC), to determine their clonal composition and differentiation potential. We will utilize DNA methylation (DNAm) profiles to determine if disease-associated epigenetic modifications are also reflected in the non-malignant counterpart.
The original cell type of CAFs will be estimated based on cell-type specific DNAm patterns. Last but not least, we will apply Epigenetic-Fibroblast-Signatures and deconvolution algorithms on the DNAm profiles to estimate the cellular composition of cancer tissue.
This research follows the hypothesis, that the percentage of CAFs can be used to stratify cancer. The epigenetic signatures will be tested on publically available DNAm datasets of various types of cancer and results will be correlated with clinical parameters, risk scores, and overall survival. Furthermore the epigenetic signatures will be prospectively analyzed in cancer tissue by barcoded bisulfite amplicon sequencing.
Our research shall provide insight into epigenetic characteristics of CAFs and may provide a new concept to use DNAm patterns of the non-malignant stromal compartment to stratify malignancies.
P04: Impact of cell cycle proteins for the crosstalk between hepatocellular carcinoma and the tumor environment
PI: Prof. Dr. Christian Liedtke
Hepatocellular carcinoma (HCC) is characterized by aberrant proliferation of hepatocytes and formation of a pro-inflammatory microenvironment containing activated hepatic stellate cells (HSC), immune cells and endothelial cells.
Our preliminary work suggests an unexpected essential role of the cell cycle protein Cyclin E1 (CcnE1) for initiation of HCC, activation and survival of HSC, and for proliferation of distinct CD45+ leukocyte populations such as macrophages.
In a translational approach, we established a pre-clinical CcnE1-targeting approach that was capable to prevent liver fibrosis progression.
In the present proposal we hypothesize that CcnE1 can trigger hepatocarcinogenesis through crosstalks of the tumor microenvironment with hepatoma cells in vivo. We aim to identify all CcnE1 expressing cell compartments in the HCC environment and the cell type specific interaction partners of CcnE1 under these conditions. We will investigate, if cell type specific ablation of CcnE1 in HSC or leukocytes can attenuate or even prevent liver cancer development.
Finally we will apply our anti-CcnE1 targeting approach in a mouse model of fibrosis-driven liver cancer. These studies will reveal if systemic or cell type specific targeting of CcnE1 could be an option for treatment of HCC.
P05: Interplay of (malignant) mast cells and mesenchymal stroma cells in CML
PI: Dr. rer. nat. Mirle Schemionek/Univ.-Prof. Dr. rer. nat. Michael Huber
Chronic myeloid leukemia (CML) is a malignant stem cell-driven disease of the hematopoietic system that is induced by the reciprocal translocation t(9;22) giving rise to the constitutively active Bcr-Abl fusion protein. A target-directed therapeutic approach inhibits activation of the Abl kinase. These tyrosine kinase inhibitors (TKI) have allowed impressive response rates, however, in the majority of CML patients malignant stem cells persist and thus TKI therapy fails to cure the malignancy. An increasing number of reports connect CML stem cell persistence with a microenvironmental support. Apparently, CML and normal microenvironments differ from each other but the mechanisms inducing these alterations remain poorly understood.
Recently, it has been shown that potent leukemic stem cells (LSCs) reflect a mast cell (MC)-associated gene signature and in vivo human CML stem cells differentiate predominantly towards aberrant MCs upon xenograft transplantation. Likewise, MCs can prime mesenchymal stromal cells (MSC) to secrete factors that in turn regulate stem cell biology.
Consequently, we aim to study the interplay of malignant MCs and stem cell persistence mediated via a CML microenvironment that is potentially primed by these MCs.
Our preliminary results show that a yet undefined cell population supports the disease.
In Aim 1, we intend to study the LSC support by normal vs malignant MSCs using our previously applied transgenic SCLtTA/Bcr-Abl mouse model. Therefore, we will co-transplant malignant MSCs isolated from CML mice together with normal or malignant stem cells. As our preliminary results suggest that MCs are increased in our mouse model, we propose the elaborate evaluation of the biology of Bcr-Abl+ CML MCs vs normal MCs (Aim 2).
Finally, transplantation of Bcr-Abl-transduced stem cells using MC-deficient Cpa3Cre/+ mice vs wildtype will unravel a potential support of this cell population in CML stem cell persistence (Aim 3). Identification of a MC axis in CML will allow for novel targeted pharmacological approaches.
P06: Role of IL6/gp130 signaling in hepatic stellate cells and its impact on HCC development in mice
PI: Dr. Tobias Otto / Univ.-Prof. Dr. Christian Trautwein
Hepatocellular carcinoma (HCC) frequently develops in a liver with chronic injury and persistent inflammation. Components of the stroma such as hepatic stellate cells (HSCs) are involved in the response to chronic liver injury and can affect HCC development by altering the tumor microenvironment. Although IL-6/gp130 signaling has been well-studied in hepatocytes, its role in HSCs and the relevance for HCC development has not been addressed. In the present proposal we want to validate our working hypothesis that IL-6/gp130 signaling in HSCs contributes to HCC development. Therefore, we aim to investigate the role of IL-6/gp130 in HSCs for the function of HSCs in vitro, as well as for the microenvironment and its impact on HCC development in vivo. We will use both loss-of-function (HSC-specific deletion of gp130) and gain-of-function (activating ligands of gp130 and HSC-specific expression of a constitutively active gp130 mutant) approaches. By employing two mouse models of chronic liver injury-induced HCC formation (CCl4/DEN and Mdr2-/-), we will be able to define the relevance of IL-6/gp130 signaling in HSC and its impact on tumor microenvironment and HCC development during chronic liver injury.
P07: PDGF signalling in myelofibrosis and myeloproliferative neoplasms
PI: PD Dr. Peter Boor / Univ.-Prof. Dr. Tim H. Brümmendorf
Platelet-Derived Growth Factor (PDGF) ligands and receptors are proto-oncogenes driving proliferation of various types of malignant cells. Different genetic alterations, e.g. fusions, mutations, translocations or amplifications, leading to increased PDGF signaling are responsible for the development of several malignancies, in particular gliomas, dermatofibrosarcoma protuberans, gastrointestinal stroma tumors and some myeloproliferative neoplasms (MPN) associated with hypereosinophilia (CMML, CML, CEL). In addition to direct effects, paracrine PDGF signaling is implicated in tumor associated angiogenesis and fibrosis. Here we aim to analyze the role of PDGF in myelofibrosis and myeloproliferative neoplasms in the following four work-packages:
(1) We aim to characterize PDGF expression and signaling in bone marrow cells using immunohistochemistry, immunofluorescence and FACS, taking advantage of specific PDGFR reporter mice.
(2) We will define the role of PDGF in stromal cells as a potential driver of myelofibrosis using transgenic mice with constitutively active PDGFR mutations activated specifically in Gli1+ stroma cells, which were shown to be crucial mediators of myelofibrosis.
(3) We will analyze the role of PDGF signaling in hematopoietic cells as a driver of myeloproliferative neoplasms using mice with constitutively active PDGFR mutations activated specifically in hematopoietic cells.
(4) Finally, using a PDGFR specific small molecule probe, we aim to explore the possibility of direct, non-invasive imaging of PDGFR in bone marrow as a direct molecular read-out in animal models outlined in WP2 and WP3.
Collectively, our data could bring novel insights into the cell-specific role of PDGF in myelofibrosis and myeloproliferative neoplasms. Our animal models could be potentially used for testing of novel treatments for these diseases. Finally, molecular PDGFR imaging could represent a novel approach to non-invasively visualize myelofibrosis.
P08: The contribution of autophagy in HSCs for the progression of HCC
PI: Dr. J. Vervoorts / Dr. M. Vucur
The tumor microenvironment cross talks with cancer cells and contributes to the development and metastasis of tumors. Cancer-associated fibroblasts (CAFs) - one major component of the tumor microenvironment - play a crucial role in proliferation, invasiveness, metastasis, and angiogenesis of cancer.
One hallmark of CAFs is the induction of autophagy helping cancer cells to overcome metabolic stress, maintain homeostasis, and support survival in a hostile microenvironment. The liver resident fibroblasts (hepatic stellate cells (HSCs)) transdifferentiate during hepatocarcinogenesis into CAFs, thereby supporting HCC development. It has been shown that HSCs are involved in HCC initiation due to a crosstalk with hepatocytes and immune cells. However, the contribution of autophagy in HSCs during HCC development remains elusive.
We identified Cyclin Y/CDK16 as a new essential regulator of autophagy in cells, but the relevant underlying molecular mechanisms are still largely unknown and the significance of our findings in animal model has not been addressed.
Activated stellate cells allow us to search for Cyclin Y/CDK16 substrates, which mediate the induction of autophagy, in cell culture.
Moreover, we will analyse the impact of disturbed autophagy in HSCs and especially validate the significance of Cyclin Y/CDK16 in HSCs in liver cancer in the well-established DEN/CCl4 model in vivo.
P09: Targeting stromal interactions in colon cancer and liver metastasis
PI: Dr. rer. nat. Wiltrud Lederle / PD Dr. rer. nat. Dr. med. Erawan Borkham-Kamphorst
Advanced colorectal cancer (CRC) is associated with a poor prognosis due to distant metastases, most frequently occurring in the liver. CRC is tightly linked to inflammation and often accompanied by a desmoplastic response. However, whereas increasing evidence indicates that fibroblasts promote colon cancer progression, the role of macrophages remains controversial, and the crosstalk between both cells types has not yet been elucidated in detail. In the present proposal, we will investigate the interplay between fibroblasts and macrophages in orthotopic metastasizing mouse colon tumors by characterizing both cell types (phenotypes, activation/ polarization state, cytokine/ chemokine signaling) during progression to liver metastasis and by therapeutic blockade of respective signaling pathways.
We will target fibroblasts by recombinant soluble PDGFRβ and deplete or block macrophages using clodronate liposomes and anti-CSF1R antibodies, respectively. In addition, we will inhibit COX-2 expressed by both cell types using low-dose aspirin and analyze the effects of the above mentioned drugs in combination. Finally, in order to investigate the clinical relevance of the data obtained in the mouse model, we will characterize human colon tumor samples (adenoma/ carcinoma at differential progression stages) for fibroblasts, macrophages and cytokine/ chemokine signaling.
The studies will provide a deeper insight into the crosstalk between fibroblasts and macrophages during colon cancer progression and may open new avenues for an improved therapy of metastatic colon cancer.
P10: The role of Nrf-2 pathways in myeloid derived suppressor cells, relevance for immunosuppression in the tumormicroenvironment?
PI: Dr. Kim Ohl / Prof. Dr. Klaus Tenbrock
Oxidative stress within the tumor microenvironment can reprogram cancer cells but also immune cells. Among Immune cells, immunosuppressive myeloid derived suppressor cells (MDSCs) play a major role in tumor immunology. MDSCs accumulate in tumors, prevent T cell mediated anti-tumor activities and are therefore a potential target for cancer therapy. Oxidative stress conditions and high levels of reactive oxygen species (ROS) activate Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the main transcriptional regulator of the anti-oxidant stress defence.
Using mice with a constitutive activation of Nrf2 in immune cells (VAVCREKeapfl/fl) we observed a dramatic expansion of splenic CD11b+Gr-1+ cells, which exhibited characteristics of MDSCs such as suppression of T cell proliferation in vitro, amelioration of T-cell mediated colitis and prevention of LPS-induced acute lung-injury in vivo.
Whole transcriptome analysis revealed Nrf2 dependent activation of cell cycle and pentose phosphate pathway. In addition, HeLa cells induce massive expansion of MDSCs in co-cultivated PBMCs, which show an upregulation of Nrf2 dependent genes. Thus oxidative stress induced Nrf2 signaling seems to be a critical transcriptional regulator for MDSC expansion.
The VAVCREKeapfl/fl mouse and the HeLa in vitro system can serve as a tool for investigation of MDSCs in cancer immunology. We will answer following questions using the above mentioned systems:
- Are Nrf2-pathways induced in human tumor associated MDSCs?
- How does Nrf2 activation in MDSCs affect intestinal tumorigenesis?
- Which genes or pathways are regulated during colitis and carcinogenesis in MDSCs?
- Do Nrf2-pathways in MDSCs affect hepatocellular carcinoma?
Our approach will uncover how oxidative stress/Nrf2 signaling influences MDSCs in tumor microenvironment and whether this serves as a potential therapeutic target.
P11: Dissecting the cross talk between megakaryocytes and Gli1+ stromal cells in bone marrow fibrosis and leukemic transformation in myeloproliferative neoplasms
PI: PD Dr. med. Rafael Kramann / Univ.-Prof. Dr. rer. nat. Martin Zenke
Bone marrow (BM) fibrosis is the continuous replacement of blood forming cells in the bone marrow by scar tissue, ultimately leading to failure of the body to produce blood cells. Primary myelofibrosis (PMF), an incurable blood cancer, is the prototypic example of the step-wise development of BM fibrosis. The specific mechanisms that cause BM fibrosis are not understood, in particular as the cells driving fibrosis have remained obscure. Our recent findings demonstrate that Gli1+ cells are fibrosis-driving cells in PMF, that their frequency correlates with fibrosis severity in patients and that their ablation ameliorates BM fibrosis. These results indicate that Gli1+ cells are the primary effector cells in BM fibrosis and that they represent a highly attractive therapeutic target. Furthermore, our data suggests that Gli1+ cells are activated by the malignant hematopoietic clone specifically by megakaryocytes through so far unknown mechanisms. Our finding puts us in a unique position to vastly expand our knowledge of the BM fibrosis pathogenesis and discover new therapeutic strategies for this fatal disease.
We will do this by:
- Dissecting the role of megakaryocytes in Gli1+ cell activation by genetic fate tracing, megakaryocyte specific gene expression and cell ablation experiments.
- We will model human PMF and megakaryocyte-stroma interaction using induced pluripotent stem cells (iPS cells) and
- We will utilize both human iPS cell disease modeling and transgenic mouse models to dissect the role of the chemokine CXCL4 in megakaryocyte-stromal interaction driving fibrosis progression and leukemic transformation.
We will apply state-of-the-art techniques, including genetic fate tracing experiments, conditional genetic knockout mouse models, iPS cell disease modeling of the bone marrow niche and CRISPR/Cas9 gene editing, to unravel the complex molecular and cellular interaction between fibrosis-causing cells and the malignant hematopoietic cells. We will translate these findings into patient samples with the aim to improve the early diagnosis and to ultimately develop novel targeted therapies with curative intentions.